analyse av muggsopp på overflater i porøse materialer og i
TRANSCRIPT
Analyse av muggsopp på overflater i porøse materialer og i luft med Mycometer-metoden
Morten Reeslev, ph.d.
Mycometer A/S,
Hørsholm, Danmark
The Technology
+
β-N-acetylhexosaminidase (NAHA)
Enzyme substrate Fluorescence
Dette enzymet er tilstede i alle muggsopp
partikler: sporer, hyfer, hyfefragmenter og
mikrofragmenter.
NAHA facts
• Tilstede i både sporer og hyfer i alle filamentøse sopper
• Enzym aktiviteten er proportional med mengden av den totale sopp biomassen i prøven.
• Korrelerer med andre sopp parametre som ergosterol ikomplekse prøver som jord og luft
• Er også funnet i ikke levende sopp partikler sommikrofragmenter
Mycometer-testen brukes for hurtigt å måle muggsopp on-site:
• på overflater
• inne i porøse materialer
• i luft
Mycometer®-test
• Mest brukte metode til måling av muggsopp i Danmark.
• Hurtigmetode, prøvetaking og analyse kan utføres på stedet på mindre enn 1 time.
• Fortolkningskategorier (de samme for alle)
• Publisert i over 20 peer reviewed artikler.
• Verifisert av US-EPA i 2011
• Brukes av flere akkrediterte laboratorier.
• Dansk inneklima standard 3033
Mycometer®-test
Måling av muggsopp på overflater.
Mycometer-surface
Muggsopp inne i porøse materialer
Måling av muggsopp i luft
•Høy variasjon i målinger over tid.
•Falske negative resultater er vanlig
•Vanskelig tolkning av resultater.
I USA har luftmålinger i lang tid værtmiskreditert.
Typisk tidsvariasjon i den målte sporekonsentrasjonen (Distribution of Spores In a Colorado Home)
Time of Sample Spore count
08:00 213
09:30 1,195
11:00 393
12:30 567
14:00 900
15:30 3,257
Source: Forensic-applications.com/moulds/sampling.html
Hvorfor denne variasjon?
Partikel størrelse (aerodynamisk diameter) µm
Tid for 1 m fald i stille luft (sek.)
1 8 timer
3 1 time
10 5,5 minutter
Unit density particles
Sopper i luften er partikler, de spres ikke veddiffusion.
Stachybotrys chartarum
aerosols are gone in about 10
minutes in stagnant air
Partikler i luften etter blåsning
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
60,00%
70,00%
80,00%
90,00%
100,00%
0 20 40 60 80 100 120 140
% a
f m
aksi
mu
m v
ærd
i
Tid i minutter
Partikler i luften efter blåsing
Enzym aktivitet
Partikler (mg/m3)
Total antal sporer
Konsentrasjonen av muggsopp i luften er bestemt av aktiviteten i rommet det måles i.
Passiv prøvetaking – før og etter aktivitet i rommene
Aggressiv prøvetaking – før og etter aktivitet
Aktiv eller aggressiv prøvetaking er tidligere blitt foreslått(Rylander, 1999).
Aggressiv prøvetaking brukes ved kvalitetskontroll etterasbest sanering (EPA guidance for clearing for reoccupancyafter asbestos decontamination) og ved prøvetaking for Anthrax (McDermott, 2004).
Aktiv/aggressiv prøvetaking
Sample# Sample ID MM-air value
per m³
A B C
1 Basement, passive sampling 206 X
2 1.st floor, passive sampling 450 X
3 2. floor, passive sampling 690 X
4 Basement, aggressive sampling 12237 X
5 1.st. floor, aggressive sampling 2493 X
6 2. floor aggressive sampling 1727 X
Passive sampling
A = MM-air value 350
B = 350 < MM-air number 450.
C = MM-air value> 450
Aggressive sampling
A = MM-air value 900
B = 900 < MM-air number 1700.
C = MM-air value> 1700
Category A: Low content of mold in the air
Category B: Medium level of mold in the air.
Category C: High level of mold in the air.
Note: These Criteria are for non-mechanically ventilated
buildings
Building: 106 Strandvejen, Roskilde Sampling date: 08/02-2010
Sampled by: Jan C. Nielsen Sampling volume: 300 L
Remarks: Both passive and aggressive sampling Case #: JCN50377
Aktiv/aggressiv prøvetaking er helt avgjørende for å fårepresentative prøver.
Jeg tror at det ved adaptering av aggressiv prøvetaking i vitenskapelige studier, vil være langt større sjanse for at man finner gode korrelasjoner mellom tilstedeværelse avmuggsopp og helseplager.
Konklusjon
Total sporetal
CFU
Total svampepartikler
CFU (KDE)= kolonidannende enheter, dvs. levende spiredyktige sporer bestemt på næringsagar. (Slit sampler + diverse) (1-10% af total sporer)
Total sporer = det totale antal igjenkjenneligesporer bestemt ved mikroskopering. Både levende og døde.(Sporetraps, filter)
Mycometer®-air = levende + dødesporer, mikropartikler samt hyfefragmenter
Total sopp-partikler = levende + dødesporer, mikropartikler samt hyfefragmenter(Ingen metode)
Hva måler forskjellige metoder?
Mycometer®-air
Enzymmålinger (Mycometer-testen) er den målemetodensom måler på det største antall partikler sammenlignetmed de metodene som er tilgjengelig i dag.
Konklusion
Traditionelt har man anvendt utendørs prøver som referanse
Sammenligning av innendørs konsentrasjon med utendørs konsentrasjon (samme Colorado Home sak)
Source: Forensic-applications.com/moulds/sampling.html
Utendørsmålinger kan ikke brukes som kvantitativreferanse for muggsopp innholdet i inneklimaet
Konklusjon
For eventuelle spørgsmål: [email protected] [email protected] (Atle Sandven) eller i vores lille bod udenfor ipauserne
Tak for jeres opmærksomhed
Der er >6 ganger så mange sporer utendørs som innendørs (CFU)
Kilde: Applied and Environmental Microbiology 2002, 68(4) 1743-1753
En undersøkelse hvor 12.026 prøver ble analysert viste:
Median inne: 80 CFU/m³
Median ute: 500 CFU/m³
How long does mold particles stay airborne?
• Theoretically, an average mold spore will fall by approx. 1 meter per hour in completely stagnant air
• It often goes a lot faster if e.g. the particles clump together or if the spores are large.– Stachybotrys chartarum aerosols are gone in about 10 minutes in
stagnant air
– Penicillium aerosols are gone in 20-30 minutes in stagnant air.
TESTING THE HYPOTHESIS
Each result given is the mean of duplicate samples.
Type of room
7:30 am
No activity prior
to sampling
14:30 pm
Activity prior to
sampling
No activity/High activity
(%)
Office, office building 22 284 8%
Music room, school 27 547 5 %
Work room, residential 73 1218 6 %
Bedroom, residential 32 2833 1 %
Work room, residential 4 425 1 %
Meeting room , office 37 154 24%
Computer room, school 8 184 4 %
Creative room, school 28 110 26 %
NAHA activity (FLU per m3) Mean 9 %
Evaluation
• In all 8 cases the level of mould was much higher in the room after activity compared to no activity.
• It is a very small study but the results are very clear and backed up by causality
Re-aerosolizing particles
• Walking/running
• Wind from an open window
• Vacuum cleaning
• Starting an HVAC system
• Starting a fan
What has been suggested to overcome the variability?
• Sampling when there has been no activity for several
hours.(it does gives much less variability, but does it
give a true representation of what level of mould particles is actually present?)
• Long term sampling (it would even out much of the variability, but what if there are no activity? If it only give one mean values then it might not be so valuable)
Alternative idea
• Creating a ”standardized activity level”
Suggested protocol for agitated sampling
Blowing on surfaces two-three times with a handheld
blower from approx. 2 meters distance. (Avoid dust
reservoirs that are not normally stirred up).
Simulating a high but naturally occurring activity
level.
Testing the protocol for Agitated/Aggressive sampling
Each result given is the mean of duplicate samples.
Type of room
7:50
No activity prior
to sampling
14:50
Activity prior to
sampling
No activity as % of
activity
Office, office building 913 1372 67%
Music room, school 929 1239 75%
Work room, residential 4927 3837 128%
Bedroom, residential 3360 3735 90%
Workroom, residential 1110 1377 80%
Meeting room , office 310 408 76%
Computer room, school 753 544 138%
Creative room, school 279 180 155%
NAHA activity (FLU per m3) Mean 103%
0
0,2
0,4
0,6
0,8
0 20 40 60 80 100 120
Tota
l du
st(m
g/m
3)
Time (minutes)
Total dust in air before and after aggressive sampling. Bedroom with non-visible mold growth.
Total Spore count before and after aggressive sampling.Bedroom with non-visible mould growth
0
500 000
1 000 000
1 500 000
2 000 000
Spo
res/
m3
40 60 80 100 120
Time in minutes
20
0
2 000
4 000
6 000
8 000
10 000
12 000
14 000
MA
V (
flo
ure
sce
nce
/m3 )
20 40 60 80 100 120
Time in minutes
Enzyme activity in air before and after aggressive sampling.Bedroom with non-visible mould growth
Thank you for your time
Fungal propagules in air
• Viable culturable spores
• Viable non culturable spores
• Non-viable spores
• Hyphal fragments (> 1 µm, Viable or non-viable)
• Microfragments (≤ 1µm)
• Konidiophorer (sporedannende enhed)
Formation of microfragments
Aggressive air sampling protocol
Windows should have been closed at least 6 hours before sampling. Note if there is
mechanical ventilation, dehumidifiers, air purifiers or the like, and if they are running.
1. Set up the pump and tripod.
2. Give the filter an ID and put it on the tube.
3. Set the timer or stopwatch to the desired time.
4. Put on the respiratory protection and set timer for 2 minutes.
5. Now blow 2-3 times on any surface from approx. 2 meter's distancewith the Makita blower. This should mimic high level of human activity e.g. vacuum cleaning or walking/running around or an open window on a windy day. Do not blow to release dust from reservoirs that are almost newer cleaned (e.g., between the lamella of a radiator).
Settling of particles after aggressive sampling
0,00%
10,00%
20,00%
30,00%
40,00%
50,00%
60,00%
70,00%
80,00%
90,00%
100,00%
0 20 40 60 80 100 120 140
% a
f m
aksi
mu
m v
ærd
i
Tid i minutter
Bedroom with no immediate visual signs of mould problems
Enzym aktivitet
Partikler (mg/m3)
Total antal sporer
Results of the study
Table 5. The table shows minimum, maximum and median Mycometer-air Values of samples collected in
both reference buildings and building with mold problems. Data from both passive and aggressive
sampling is shown. n = 35.
Passive sampling
Reference buildings Mold problem buildings
(all rooms)
Mold problems buildings
(only rooms with mold
source)
Minimum – maximum 57-880 57–10723 167-10723
Median value (all rooms) 193 412 462
Aggressive sampling
Minimum – maximum 113-2410 217-76233 707-76233
Median value 428 1468 4013
A
MM-air numbers 900 Low level of mold in the air.
The level of mold in the air is like that found in normal buildings with normal cleaning standards.
Even with an A response and thus good air quality, it can not be excluded that there may be
mold attack hidden in a building construction.
B
900< MM-air numbers 1700 Medium level of mold in the air.
The level of mold is higher than that found in normal buildings with normal cleaning standard.
This may be due to accumulation of exogenous mold that accumulates due to poor cleaning
standards.
A category B result should always give rise to a more thorough inspection to look for a mold
source.
C
MM-air numbers > 1700 High level of mold in the air.
The level of mold is significantly higher than that found in normal buildings. This may be because
there is a source of mold in the room / building. A source may be growth of mold on / in
buildings; but it can also be growth in firewood, potted plants, rotten fruit / vegetables,
garbage, etc. Finally, a very poor cleaning standard could leave a large accumulation of external
mold particles, that, for example by activity, can swirl up into the air.
A
MM-air numbers 350 Low level of mold in the air..
The level of mold in the air is like that found in normal buildings with normal cleaning standards.
Even with an A response and thus good air quality, it can not be excluded that there may be mold
attack hidden in a building construction.
B
350< MM-air numbers 450 Medium level of mold in the air.
The level of mold is higher than that found in normal buildings with normal cleaning standard. This
may be due to accumulation of exogenous mold that accumulates due to poor cleaning standards.
A category B result should always give rise to a more thorough inspection to look for a mold
source.
C
MM-air numbers > 450 High level of mold in the air.
The level of mold is significantly higher than that found in normal buildings. This may be because
there is a source of mold in the room / building. A source may be growth of mold on / in buildings;
but it can also be growth in firewood, potted plants, rotten fruit / vegetables, garbage, etc.
Finally, a very poor cleaning standard could leave a large accumulation of external mold particles,
that, for example by activity, can swirl up into the air.
Publications• The Use of Fluorogenic Substrates to Measure Fungal Presence and
Activity in Soil. Appl. Environ. Microbiol. 64:613-617. M. Miller, A. Palojärvi, A. Rangger, M. Reeslev, A. Kjøller. 1998.
• Quantifying Mold Biomass on Gypsum Board: Comparison of Ergosteroland Beta-N-Acetylhexosaminidase as Mold Biomass Parameters. Applied and Environmental Microbiology. Vol. 69, No.7, p. 3996-3998. M.Reeslev, M.Miller, KF Nielsen. 2003.
• Analytical Instrument Performance Criteria: Application of a Fluorometric Method for the Detection of Mold in Indoor Environments. Applied Occupational and Environmental Hygiene. Vol. 18, No.7, p. 499-503. D Krause, YY Hamad, L Ball. 2003.
• The Mycometer™-Test: A New Rapid Method For Detection And Quantification Of Mold In Buildings.Proceedings of Healthy Buildings 2000, Vol. 1, p.589-590. M.Reeslev and M. Miller. 2000.
• )
Publications• Nagase Activity In Airborne Biomass Dust And Relationship Between
Nagase Concentrations And Fungal Spores. Aerobiologia Vol. 19, 97 – 105. A.M., Madsen. 2003.
• Application of a Fluorometric Method for the Detection of Mold in Indoor Environments. (2003), D.Krause. Applied Occupational and Environmental Hygiene Volume 18(7): 1–5.
• Successful Mold Growth Remediation in HVAC Systems. P Buckmaster. Occupational Health and Safety, January 2008.
• Airborne enzyme measurements to detect indoor mould exposure. Journal of Environmental Monitoring, V.12, p.2161–2164. R. Rylander, et al. 2010
• Fluorometric detection and estimation of fungal biomass on cultural heritage materials. Journal of Microbiological Methods 80 (2010) 178–182, R Mitchell, et al ( Harvard) 2010
Publications
• Beyond LEED, Pre and Post Occupancy Evaluations for New Buildings. P Buckmaster. Synergist, May 2011.
• Aggressive Sampling, Improving the Predictive Value of Air Sampling For Fungal Aerosols. M. Reeslev, M. Miller, JC Nielsen, L Rogers. Proceedings of Indoor Air Conference, ISIAQC. June 2011, Austin Texas.
• Airborne enzyme measurements for the identification of mouldy buildings. Rylander R, Reeslev M, Hulander T. . J Environ Monit, 2010; 12:2161-2164
• Airborne enzyme in homes of patients with sarcoidosis. Terčelj M, Salobir B, Rylander R. Env Health 2011; 10; 8-13.
• Nocturnal asthma and domestic exposure to fungi. Terčelj M, Salobir B, Narancsik Z, Kriznar K, Grzetic-Romcevic T, Matos T, Rylander R. Indoor + Built Env 2012; submitted.